PPARα Agonists and TGFβ Inhibitors Stimulate Red Blood Cell Production by Enhancing Self-Renewal of BFU-E Erythroid Progenitors

Abstract

The production of red blood cells (RBCs) in mammals is tightly regulated by erythropoietin (Epo), which stimulates erythropoiesis by promoting survival, proliferation, and terminal differentiation of the colony-forming unit erythroid progenitors (CFU-Es). Several acute and chronic anemias, including hemolysis, severe trauma-induced anemia and genetic bone marrow failure disorders such as Diamond-Blackfan anemia (DBA), are not treatable with Epo because the CFU-E erythroid progenitors that respond to Epo are either too few in number or are not sensitive enough to Epo to maintain adequate RBC production. Treatment of Epo-resistant anemias requires a drug that acts earlier than Epo in erythropoiesis and that enhances the formation of CFU-Es. One attractive approach is to devise strategies to promote self-renewal of the upstream burst-forming unit erythroid progenitors (BFU-Es). We recently showed that, like transient amplifying cells in other stem-cell rooted developmental pathways, BFU-Es can undergo a limited number of self- renewal divisions before entering a differentiation pathway leading to formation of CFU-Es. Over time BFU-E self-renewal increases the number of CFU-Es and, after ~2-3 additional days, results in increased numbers of reticulocytes produced from each initiating BFU-E. We showed that glucocorticoids increase the probability that a BFU-E undergoes a self-renewal division and over time increase the numbers of red cells formed from a BFU-E. Recently we showed that activation of two other nuclear receptor/transcription factors - PPARα (by the lipid - lowering drugs fenofibrate and GW7647) and HIF1α (by two clinically- tested Prolyl Hydroxylase 2 (PH2) inhibitors) synergize with low corticosteroid concentrations to further enhance BFU-E self-renewal and red cell production. "Early" BFU-E cells forming large BFU-E colonies presumably have higher capacities for self-renewal than do those forming small BFU-E colonies. In order to understand the mechanism underlying this heterogeneity, we conducted single cell transcriptome analysis on BFU-E cells purified from mouse embryos. Our analyses showed that there are two principal subgroups of mouse BFU-E cells and that expression of the Type III TGFβ receptor (TGFβ RIII) is markedly elevated in "late" relative to "early" BFU-Es. Expression of TGFβ RIII is correlated with that of GATA1, a gene encoding an erythroid transcription factor induced during the BFU-E to CFU-E transition. Both mouse and human BFU-E sub populations (TGFBR310%lo) expressing the 10% lowest amount of surface TGFβ RIII are indeed enriched for early BFU-Es, and are significantly more responsive to glucocorticoid stimulation, which promotes BFU-E self-renewal, as compared to the total BFU-E population. The TGFBR310%lo BFU-E subpopulation presumably represents earlier BFU-Es with maximal capacity for self-renewal. Consistent with this notion, signaling by the TGFβ receptor kinases RI and RII increases during the transition from early (TGFBR310%low) to late (TGFBR310%hi) BFU-Es and then decreases in CFU-E cells. Blocking TGFβ signaling by receptor kinase inhibitors increases TGFBR310%lo BFU-E cell self-renewal and increases total erythroblast production, suggesting the use of this type of drug in treating EPO unresponsive anemias. Disclosures No relevant conflicts of interest to declare.